1. Field of the Invention
The present invention concerns a novel β-secretase, a method of partially purifying this novel β-secretase, and its use in assays to screen for potential drug candidates against Alzheimer's disease and other neurological diseases.
2. Description of the Related Art
A number of important neurological diseases, including Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), and prion-mediated diseases are characterized by the deposition of aggregated proteins, referred to amyloid, in the central nervous system (CNS) (for reviews, see Glenner et al., J. Neurol. Sci. 94:1–28 [1989]; Haan et al, Clin. Neurol. Neurosurg. 92(4):305–310 [1990]). These highly insoluble aggregates are composed of nonbranching, fibrillar proteins with the common characteristic of β-pleated sheet conformation. In the CNS, amyloid can be present in cerebral and meningeal blood vessels (cerebrovascular deposits) and in the brain parenchyma (plaques). Neuropathological studies in human and animal models indicate that cells proximal to amyloid deposits are disturbed in their normal functions (Mandybur, Acta Neuropathol. 78:329–331 [1989]; Kawai et al., Brain Res. 623:142–146 [1993]; Martin et al., Am. J. Pathol. 145:1348–1381 [1994]; Kalaria et al., Nuroreport 6:477–480 [1995]; Masliah et al., J. Neurosci. 16:5795–5811 [1996]; Selkoe, J. Biol. Chem. 271:18295–18298 [1996]; Hardy, Trends Neurosci 20:154–159 [1997]).
AD and CAA share biochemical and neuropathological markers, but differ somewhat in the extent and location of amyloid deposits as well as in the symptoms exhibited by affected individuals. The neurodegenerative process of AD, the most common neurodegenerative disorder worldwide, is characterized by the progressive and irreversible deafferentation of the limbic system, association neocortex, and basal forebrain accompanied by neuritic plaque and tangle formation (for a review, see Terry et al., “Structural alteration in Alzheimer's disease,” In: Alzheimer's disease, Terry et al. Eds., 1994, pp. 179–196, Raven Press, New York). Dystrophic neurites, as well as reactive astocytes and microglia, are associated with these amyloid-associated neuritic plaques. Although the neuritic population in any given plaques is mixed, the plaques generally are composed of spherical neurites that contain synaptic proteins, APP (type I), and fusiform neurites containing cytoskeletal proteins and paired helical filaments (PHF; type II).
CAA patients display various vascular syndromes, of which the most documented is cerebral parenchymal hemorrhage. Cerebral parenchymal hemorrhage is the result of extensive amyloid deposition within cerebral vessels (Hardy, Trends Neurosci 20:154–159 [1997]; Haan et al., Clin. Neurol. Neurosurg. 92:305–310 [1990]; Terry et al., supra; Vinters, Stroke 18:211–224 [1987]; Itoh et al., J. Neurosurgical Sci. 116:135–141 [1993]; Yamada et al., J. Neurol. Neruosurg. Psychiatry 56:543–547 [1993]; Greenberg et al., Neurology 43:2073–2079 [1993]; Levy et al., Science 248:1124–1126 [1990]). In some familial CAA cases, dementia was noted before the onset of hemorrhages, suggesting the possibility that cerebrovascular amyloid deposits may also interfere with cognitive functions.
Both AD and CAA are characterized by the accumulation of senile plaques in the brains of the affected individuals. The main amyloid components is the amyloid β protein (Aβ), also referred to as amyloid β or β-amyloid peptide, derived from proteolytic processing of the β-amyloid precursor protein, β-APP or simply APP. For review in connection with AD see, Selkoe, D. J. Nature 399: A23–A31 (1999). Aβ is produced by proteolytic cleavage of an integral membrane protein, termed the β-amyloid precursor protein (βAPP).
The Aβ peptide, which is generated from APP by two putative secretases, is present at low levels in the normal CNS and blood. Two major variants, Aβ1-40 and Aβ1-42 are produced by alternative carboxy-terminal truncation of APP (Selkoe et al. (1988) Proc. Natl. Acad. Sci. USA 85:7341–7345; Selkoe (1993) Trends Neurosci 16:403–409). Aβ1-42 is the more fibrillogenic and more abundant of the two peptides in amyloid deposits of both AD and CAA. In addition to the amyloid deposits in AD cases described above, most AD cases are also associated with amyloid deposition in the vascular walls (Hardy (1997), supra; Haan et al. (1990), supra; Terry et al., supra; Vinters (1987), supra; Itoh, et al. (1993), supra; Yamada et al. (1993); supra; Greenberg et al. (1993), supra; Levy et al. (1990), supra). These vascular lesions are the hallmark of CAA, which can exist in the absence of AD.
The precise mechanisms by which neuritic plaques are formed and the relationship of plaque formation to the AD-associated, and CAA-associated neurodegenerative processes are not well-defined. However, evidence indicates that dysregulated expression and/or processing of APP gene products or derivatives of these gene products are involved in the pathophysiological process leading to neurodegeneration and plaque formation. For example, missense mutations in APP are tightly linked to autosomal dominant forms of AD (Hardy (1994) Clin. Geriatr. Med. 10:239–247; Mann et al. (1992) Neurodegeneration 1:201–215). The role of APP in neurodegenerative diseases is further implicated by the observation that persons with Down's syndrome who carry an additional copy of the human APP (hAPP) gene on their third chromosome 21 show an overexpression of hAPP (Goodison et al. (1993) J. Neuropathol. Exp. Neurol. 52:192–198; Oyama, et al. (1994) J. Neurochem. 62:1062–1066) as well as a prominent tendency to develop AD-type pathology early in life (Wisniewski et al. (1985) Ann. Neurol. 17:278–282). Mutations in Aβ are linked to CAA associated with hereditary cerebral hemorrhage with amyloidosis (Dutch HCHWA) (Levy, et al. (1990), supra), in which amyloid deposits preferentially occur in the cerebrovascular wall with some occurrence of diffuse plaques (Maat-Schieman et al. (1994) Acta Neuropathol. 88:371–8; Wartendorff et al. (1995) J. Neurol. Neurosurg. Psychiatry 58:699–705). A number of hAPP point mutations that are tightly associated with the development of familial AD encode amino acid changes close to either side of the Aβ peptide (for a review, see, e.g., Lannfelt et al. (1994) Biochem. Soc. Trans. 22:176–179; Clark et al. (1993) Arch. Neurol. 50:1164–1172). Finally, in vitro studies indicate that aggregated Aβ can induce neurodegeneration (see, e.g., Pike et al. (1995) J. Neurochem. 64:253–265).
APP is a glycosylated, single-membrane-spanning protein expressed in a wide variety of cells in many mammalian tissues. Examples of specific isotypes of APP which are currently known, to exist in humans are the 695-amino acid polypeptide described by Kang et al. (1987) Nature 325:733–736, which is designated as the “normal” APP. A 751-amino acid polypeptide has been described by Ponte et al. (1988) Nature 331:525–527 and Tanzi et al. (1988) Nature 331:528–530. A 770-amino acid isotype of APP is described in Kitaguchi et al. (1988) Nature 331:530–532. A number of specific variants of APP have also been described having mutations which can differ in both position and phenotype. A general review of such mutations is pivoted in Hardy (1992) Nature Genet. 1:233–235. A mutation of particular interest is designated the “Swedish” mutation where the normal Lys-Met residues at positions 595 and 596 are replaced by Asn-Leu. This mutation is located directly upstream of the normal β-secretase cleavage site of APP, which occurs between residues 596 and 597 of the 695 isotype.
APP is post-translationally processed by several proteolytic pathways resulting in the secretion of various fragments or intracellular fragmentation and degradation. F. Checler, J. Neurochem. 65:1431–1444 (1995). The combined activity of β-secretase and γ-secretase on APP releases an intact β-amyloid peptide (Aβ), which is a major constituent of amyloid plaques. Aβ is an approximately 43 amino acid peptide which comprises residues 597–640 of the 695 amino acid isotype of APP. Internal cleavage of Aβ by a α-secretase inhibits the release of the full-length Aβ peptide. Although the extent of pathogenic involvement of the secretases in AD progression is not fully elucidated, these proteolytic events are known to either promote or inhibit Aβ formation, and thus are thought to be good therapeutic candidates for AD.
There are at least two proteases involved in the generation of Aβ, referred to as α- and γ-secretases (Citron et al., Neuron 17:171–179 [1996]; Seubert et al., Nature 361:260–263 [1993]; Cai et al., Science 259:514–516 [1993]; and Citron et al., Neuron 14:661–670 [1995]). There have been intense efforts in recent years to identify and characterize these enzymes. Recently five independent groups have reported cloning and characterization of genes corresponding to β-secretase (Vassar et al., Science 286: 735–741 [1999]; Yan et al., Nature 402: 533–537 [1999]; Sinha et al., Nature 402: 537–540 [1999]; Hussain et al., Mol. Cell. Neurosci. 14: 419–427 [1999]; Lin et al. Proc. Natl. Acad. Sci. USA 97: 1456–1460 [2000]). The enzyme has been variously referred to as β-site APP-cleaving enzyme (BACE), Aspartyl protease-2 (Asp2), memapsin 2 or simply as β-secretase. However, the deduced amino acid sequence of the polypeptide chain reported by all four groups is identical. The cloned enzyme possesses many of the characteristics expected of an authentic β-secretase, such as the presence of conserved aspartyl protease active site motif (D[S/T]G), a signal peptide, a transmembrane domain, ability to act upon βAPP, enhanced cleavage of the Swedish mutant of βAPP, and intracellular localization in Golgi, endoplasmic reticulum, endosome/lysosome compartments. Thus, it appears that the newly identified enzyme represents an authentic β-secretase. However, none of these reports rule out the possibility of additional enzymes having β-secretase activities. There have been speculations about the possible involvement of more than one enzyme entity in β-cleavage of APP (e.g., Papastoitsis et al. Biochem. 33: 192–199 [1994]; Koike et al. J. Biochem. 126: 235–242 [1999]; Brown et al. J. Neurochem. 66: 2436–2445 [1996]; Chevallier et al. Brain Res. 750: 11–19 [1997]). Experiments with intact cells suggested that β-secretase has an acidic pH optimum (Haass et al., J. Biol. Chem. 268: 3021 [1993]; Knops et al., J. Biol. Chem. 270: 2419 [1995]). Indeed, the newly isolated and characterized β-secretase was found to have a pH optimum at 4.5 (Vassar et al., Science 286: 735–741 [1999]) or 5.0 (Sinha et al., Nature 402: 537–540 [1999]) for its activity. These characteristics are thought to be consistent with β-secretase activity in the endosomes/lysosomes where the pH is low. However, β-secretase is also known to function in the endoplasmic reticulum where the pH is neutral (around 7.0), an environment not well suited for an enzyme having an acidic pH optimum. It is, therefore, possible that there are more than one β-secretase enzymes involved in the proteolytic cleavage of β-APP.
The identification, isolation, purification, and characterization of all participants in the enzymatic cleavage of β-APP would permit chemical modeling of a critical event in the pathology of Alzheimer's disease and would allow the screening of compounds to determine their ability to inhibit in vivo β-secretase activity. For these reasons, it would be desirable to isolate, clone and characterize a β-secretase enzyme having a neutral pH preference. It would be also desirable to develop a method for the functional cloning of the genes encoding proteins or enzymes involved in the proteolytic cleavage of β-APP. It would further be desirable to identify inhibitors of the β-APP processing leading to Aβ release, and in particular to identify molecules that preferentially inhibit the β-secretase activity having a neutral (pH 6.5–7.0) pH optimum, or the β-secretase activity having an acidic (around pH 4.5) pH optimum.